Human papillomaviruses (HPV) are the causative agents of cervical, anal and many oral cancers. HPVs infect stratified squamous epithelia and link their productive life cycles to the differentiation of the infected cell. A low level of viral gene expression is detected in infected basal cells and significant levels of viral transcripts are only seen in differentiated cells. Productive genome replication, late gene expression, and virion assembly are restricted to the nuclei of highly differentiated cells present in the uppermost epithelial layers. The goal of our studies is to understand how HPV gene expression along with the viral life cycle is controlled by cellular transcription factors. My laboratory recently demonstrated that the amplification of viral genomes in differentiating cells is dependent on the constitutive activation of the ATM double strand DNA break repair pathway in HPV positive cells. Our studies have shown that this results from E7-induced activation of the innate immune transcription factor, STAT-5. pSTAT-5 then activates ATM through its effects on the acetyltransferase Tip60. STAT-5 also turns on the ATR single strand DNA damage repair pathway by directly increasing the transcription of the replication/transcription factor TopBP1 that is a binding partner of ATR. For genome amplification to occur in suprabasal layers the ability of cells to re-enter S/G2 must be retained and our studies show that is dependent on p63 as well as the downstream transcriptional activator KLF4 that are controlled through HPV-induced post-translational effects. In HPV positive cells, KLF4 was found to regulate the expression of a number of unique targets as well as to form complexes with one of its targets, Blimp1, to directly activate late viral expression. Another important regulator is the insulator factor CTCF that we have shown binds to sequences in the late regions of HPV 31 to regulate viral expression as well as replication. Similar CTCF binding motifs are found in the late regions of all HPV types. We also observed that HPVs alter the expression of microRNAs to regulate the differentiation-dependent late phase of the viral life cycle. Our studies show that HPV mediated repression of miR-145 is important for regulating KLF4 levels while suppression of miR-424 is important for increasing levels of the ATR downstream kinase CHK1. The studies proposed in this renewal application build on these observations and will examine how viral expression during the life cycle is regulated by changes in chromatin, DNA lopping and differentiation-induced transcription factors. Aim 1: How do changes in methylation of histones bound to HPV episomes regulate activation of the late viral promoter during the differentiation dependent life cycle? Aim 2: Do CTCF/SMC1 regulate viral expression through DNA looping to other viral genomes or cellular sites? Aim 3: How do KLF4/Blimp1 and CEBP??cooperate to activate late expression? Are histone methyltransferase or demethylases associated with these complexes? Are novel factors also detected in these complexes?